This invention relates to a method of extracting nucleic acids from a sample. More particularly, this invention relates to a method of nucleic acid extraction and a method of specified nucleic acid sequence detection that can be used in biotechnology and clinical diagnosis.
Extracting nucleic acids from a sample is an important operation in biotechnology, clinical diagnosis and other sophisticated fields. For instance, gene recombinant technology requires the isolation of both a vector DNA and the DNA to be cloned and, in order to make a gene assay on genetic diseases and cancer genes, it is necessary to extract nucleic acids from leukocyte cells and the like present in blood.
Nucleic acids generally do not occur as free molecules but rather in bacteria, cells, virus particles, etc. as they are covered with cell membranes and walls which are composed of proteins, lipids and sugars. Nucleic acids themselves form complexes with histone and other proteins. To extract nucleic acids which are present in this manner, the cell membranes and walls covering them must be disrupted and the proteins of the complexes mentioned above denatured or degraded to thereby become soluble, so that the nucleic acids are freed and then extracted.
Nucleic acids are conventionally extracted by one of the following methods:
(i) the so-called proteinase K/phenol method, in which a proteolytic enzyme such as proteinase K or a surfactant is added to disrupt the cell membrane or wall and the protein of a complex of interest is degraded to free nucleic acids; then phenol/chloroform are added and the mixture is centrifuged to have the nucleic acids transferred into the aqueous phase; the aqueous phase is recovered by separation and ethanol, isopropanol or the like is added to the recovered aqueous phase, thereby rendering the nucleic acids insoluble (Molecular Cloning: A Laboratory Manual, Appendices E3-E4, New York, Cold Spring Harbor Laboratory, 1989); PA1 (ii) the so-called AGPC method, in which a liquid mixture of guanidinium isothiocyanate and phenol is added to a sample of interest to disrupt the cell membrane and wall, so that the protein of the complex is denatured to become soluble; nucleic acids are then freed and chloroform is added to transfer the nucleic acids to the aqueous phase; the aqueous phase is recovered by separation and thereafter, ethanol, isopropanol or the like is added to the recovered aqueous phase, thereby rendering the nucleic acids insoluble (Acid Guanidinium-Thiocyanate Phenol-Chloroform Method: Analytical Biochemistry, 162, 156-159, 1987); PA1 (iii) the so-called guanidinium method, in which guanidinium hydrochloride or guanidinium thiocyanate is added to a sample of interest to disrupt the cell membrane and wall, so that the protein of the complex is denatured to become soluble; nucleic acids are then freed and ethanol or the like is added to render the free nucleic acids insoluble (Molecular Cloning: A laboratory Manual, 7.23-7.25, New York, Cold Spring Harbor Laboratory, 1989; and Analytical Biochemistry 162, 463, 1987); and PA1 (iv) the so-called sodium iodide method, in which sodium iodide containing glycogen which has affinity for the nucleic acid to be extracted is added to a sample of interest, whereby the cell membrane and wall are disrupted and the protein of the complex is denatured, so that it becomes soluble; nucleic acids are then freed and isopropanol is added to render the free nucleic acids and glycogen insoluble (Nucleic Acid Res., 19 (20), 592, 1991). PA1 (1) mixing the sample with a carrier which is at least one member selected from the group consisting of dextran, acrylamide and carboxymethyl cellulose to form a liquid mixture; PA1 (2) mixing said liquid mixture with reagent C to render the nucleic acids and the carrier insoluble, said reagent C containing at least one reagent A selected from the group consisting of guanidinium thiocyanate, guanidinium hydrochloride, potassium thiocyanate and sodium thiocyanate and at least one reagent B selected from the group consisting of n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, sec-butyl alcohol, tert-butyl alcohol and tert-amyl alcohol; and PA1 (3) separating the insolubilized nucleic acids and carrier from the liquid phase. PA1 (1) mixing a sample with a carrier which is at least one member selected from the group consisting of dextran, acrylamide and carboxymethyl cellulose to form a liquid mixture; PA1 (2) mixing said liquid mixture with reagent C to render the nucleic acids and the carrier insoluble, said reagent C containing at least one reagent A selected from the group consisting of guanidinium thiocyanate, guanidinium hydrochloride, potassium thiocyanate and sodium thiocyanate and at least one reagent B selected from the group consisting of n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, sec-butyl alcohol, tert-butyl alcohol and tert-amyl alcohol; PA1 (3) separating the insolubilized nucleic acids and carrier from the liquid phase; PA1 (4) converting the separated nucleic acids to DNA by a reverse transcription reaction if they are RNAs; PA1 (5) subjecting the nucleic acids, either separated in step (3) or obtained in step (4), to a PCR reaction in a polymerase chain reaction solution that contains an oligonucleotide probe suitable for amplifying at least a specified sequence, a mononucleotide triphosphate mixture, a polymerase and an intercalating fluorochrome; PA1 (6) measuring the change that occurs in the intensity of fluorescence as a result of the PCR reaction or the change that occurs in the intensity of fluorescence from the reaction solution during the PCR reaction; and PA1 (7) determining, on the basis of the change in the intensity of fluorescence, whether a nucleic acid having the specified sequence was present in the sample. PA1 (1) at least one carrier selected from the group consisting of dextran, acrylamide and carboxymethyl cellulose; and PA1 (2) reagent C containing at least one reagent A selected from the group consisting of guanidinium thiocyanate, guanidinium hydrochloride, potassium thiocyanate and sodium thiocyanate and at least one reagent B selected from the group consisting of n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, sec-butyl alcohol, tert-butyl alcohol and tert-amyl alcohol.
The four methods described above have their own disadvantages. The proteinase K/phenol method involves complicated procedures since it takes much time to accomplish degradation with the proteolytic enzyme and because temperature control is necessary to ensure an effective enzyme reaction. Furthermore, phenol and chloroform are toxic chemical reagents (both of them are designated as deleterious substances in the Poisonous and Deleterious Substances Control Law) and to handle them, the use of protective clothes, gloves, gas proof hoods, etc. is necessary. Furthermore, the liquid waste resulting from the extracting procedure must be subjected to a special treatment, requiring extra cost, time and facilities. In addition, great skill is required to recover by separation the aqueous phase into which nucleic acids of interest have been transferred and this makes it difficult to achieve consistent efficiency in the extraction of nucleic acids.
Similar problems occur in the AGPC method in connection with the handling of phenol and chloroform, as well as in the efficiency of extraction that is related to the recovery by separation of the aqueous phase into which nucleic acids of interest have been transferred. In addition, the AGPC method is specifically intended for extracting RNA and unsuitable for extraction of DNA.
In the guanidinium method, ethanol or the like is added to a sample of interest after the addition of guanidinium hydrochloride or the like. In order to prevent a subsequent drop in the concentration of guanidinium in a solution, highly concentrated (ca. 6-8 M) guanidinium must be used but then the chances of a guanidinium salt being deposited will increase (particularly in winter or when room temperature is no more than 20.degree. C.). The deposition of a guanidinium salt may lead to the plugging of pipettes and other equipment used in the addition of guanidinium hydrochloride or the like and this presents a substantial obstacle to any attempt to mechanize the procedure of the guanidinium method. Ethanol or the like is added subsequent to guanidinium hydrochloride, etc. and to render the nucleic acids insoluble, the final concentration of ethanol or the like must be 50-70%. However, the volume of the sample is increased on account of the addition of guanidinium hydrochloride or the like and, hence, even ethanol of high purity (ca. 100%) must be used in an amount at least equal to the liquid mixture of the sample and guanidinium hydrochloride or the like.
The sodium iodide method is subject to a problem in that it requires incubation at 60.degree. C. for solubilizing proteins or the like, which results in a low efficiency of RNA extraction. Another problem is that labile iodide ion (I.sup.-) tends to be oxidized by the action of light or the like and to form an iodine molecule (I.sub.2). To avoid this, the reagents have to be stored in a cold dark room.
The polymerase chain reaction (PCR) is known as a method of DNA amplification (Molecular Cloning: A Laboratory Manual, Chap. 14, New York, Cold Spring Harbor Laboratory, 1989) that is capable of gene assay (genetic diagnosis) with ultra-high sensitivity. If DNA amplification is successful, PCR has a potential to accomplish amplification by a factor of at least 100 million. However, in view of the possibility for amplification of even one molecule of DNA, the assay result will be adversely affected in the presence of a DNA (exogenous DNA) that should be absent from the PCR nucleic acid sample or the PCR reagent.
One of the causes for the presence of exogenous DNA occurs at the stage where nucleic acids are extracted from the sample of interest to prepare the PCR nucleic acid sample and this also concerns the aerosol that is generated in the procedure of extracting the nucleic acids of interest. The generation of the aerosol is highly likely to occur in the proteinase K/phenol method or the AGPC method since besides the step of adding the reagent to the sample and performing other procedures such as mixing, these methods also require time-consuming procedures such as recovering the aqueous phase by separation. The particles of the aerosol have a diameter of about 20 .mu.m and their volume is only about 4.times.10.sup.-6 .mu.L. Consider, for example, a patient suffering from hepatitis B; since ca. 10.sup.5 virus particles can be contained in 1 .mu.L of blood, one aerosol particle can contain one virus particle. In other words, the aerosol can do serious harm to PCR which is capable of amplifying one molecule of DNA. Therefore, there is a need to reduce the number of treatment steps involved.
In order to minimize the generation of the aerosol in a PCR process, a method has been proposed in European Patent Publication No. 487,218 that a PCR reaction can be carried out in a harmetic state by using an intercalating fluorescent dye (fluorochrome) or the like to quantify a target DNA in a sample. However, the difficulty to minimize the generation of aerosol in an extraction process of nucleic acid from a sample is still remaining.